Most screening systems currently used to identify potential antineoplastic drugs evaluate the ability of compounds to kill rapidly-growing cells in culture. Drugs identified in such systems are thus generally not specific for tumor cells but are also toxic to rapidly-growing normal cells in the body. Out of more than 400,000 compounds that have been evaluated in such systems, fewer than twenty have shown an acceptably low level of toxicity, and even these compounds show toxic effects in most cancer patients.
More effective cancer chemotherapy will require the identification of new drugs that act to specifically kill cancer cells or to suppress the transformed phenotype, while exhibiting low toxicity to normal cells. To find these new drugs, new screening systems will be required.
Our understanding of the molecular basis of cancer has been revolutionized by the identification of a relatively small set of normal cellular genes called protooncogenes which, when altered, can produce neoplastic change Bishop, Ann. Rev. Biochem. 52:301 (1983); Varmus, Ann. Rev. Genetics 18:553 (1984)!. Alterations in protooncogene expression can occur for a variety of reasons, including mutations, nucleotide substitutions, chromosomal translocations, gene amplifications, and the insertion of mobile genetic elements. As a result of such changes, the expression of protooncogenes may be altered or they may be mutated to encode altered protein products.
The proteins encoded by protooncogenes play an important role in governing many aspects of cell growth and development. Mutant or activated protooncogenes are believed to make specific contributions to the phenotypes of tumor cells and hence are called oncogenes.
One of the remarkable features of cellular protooncogenes is that they have shown extraordinary conservation during evolution. Several of these genes have been identified in organisms as diverse as yeast, mammals, birds, fish and insects. This evolutionary conservation suggests that the proteins encoded by the protooncogenes must have important functions in normal cell growth and development, with each directing a particular event in the complex system of signals that regulates the proliferation and differentiation of cells. Changes in any one or more of these genes can lead to cancer (Bishop, supra; Varmus, supra).
The proteins encoded by protooncogenes fall into several groups. Some are growth factors--polypeptides that signal cells to divide. Others are receptors for growth factors, molecules that are embedded in the cell membranes and respond to growth factors. Another group is known as the group of G proteins, which transmit signals from receptors to other components of the signal-transduction pathway. Others are protein kinases which phosphorylate other proteins. Still others are nuclear proteins that are involved in DNA transcription Weinberg, Science 230:770 (1985)!. A schematic representation of the pathway by which signals generated outside a cell can transmit information to the nucleus to produce cell division is shown in FIG. 1.
More recent research has focused on the part played by negative regulators of cell growth in the development of cancer. These negative regulators are known as tumor suppressor genes (also known as recessive oncogenes or anti-oncogenes). Unlike oncogenes of viral and cellular origin, which appear to act in a dominant manner to confer transformed characteristics, loss of both copies of these recessive oncogenes is required for neoplastic change Stanbridge, Bioessays 3:252 (1985)!.
A protein called the Rb protein which is encoded by one such anti-oncogene, the retinoblastoma anti-oncogene, is presumed to act in the control of the cell cycle. Oncogenes carried by DNA tumor viruses such as SV-40 large T antigen and adenovirus EIA function by complexing with and inactivating the Rb protein Whyte, et al., Nature 334:124 (1988)!.
Although oncogenes have been linked to tumor growth, the signalling pathways controlled by oncogene proteins are not limited to growth control alone. Oncogene-encoded proteins probably regulate other biological activities such as transmission of nerve impulses, phototransduction, chemotaxis, differentiation, etc. Alterations in pathways controlling such activities may play an important role in other diseases such as atherosclerosis and Alzheimer's disease. Hence, specific drugs designed to inhibit the activities regulated by mutant oncogene proteins may prove useful in the treatment not only of cancer, but of many other diseases as well.
When rat embryo fibroblasts undergo neoplastic transformation, microfilaments containing actins and myosins are reorganized from a bundle state into a randomly interwoven meshwork Pollack et al., Proc. Natl. Acad. Sci. USA 72:994 (1975)!. This phenomenon, known as actin cable network diffusion, has been found to be a common characteristic of many such transformed cells. Studies indicate that changes in different cytoskeletal components are not an indirect consequence of transformation but are specific to the oncogenes that cause transformation Franza et al., Cancer Cells 1:137 (1984); Leavit, J., in Human Fibroblast Transformation (Ed., G. Milo), CRC Press Inc., 1989, pp. 1-28!.
Changes in the arrangement of cytoskeletal components have been associated with alterations in cell growth rate, attachment, saturation density and the expression of the differentiated phenotype. Such changes may favor neoplastic growth and play an important role in tumor initiation or progression. Although detailed understanding of the molecular mechanisms involved in these cytoskeletal changes is lacking, it is clear that some genes which are silent in normal cells are turned on in transformed cells, and that certain others that are expressed in normal cells are turned off following transformation.
Studies by Leavitt et al. Nature 316:840 (1985)! and Garrels et al. Cancer Cells 1:137 (1984)! have shown that smooth muscle .alpha.-actin isoform is expressed in both Rat2 and REF52 cells and is repressed following neoplastic transformation of the cells by several RNA and DNA tumor viruses.
Investigations on the human smooth muscle myosin light chain-2 (MLC-2) isoform have shown that the MLC-2 gene also is specifically repressed when fibroblasts undergo neoplastic transformation Kumar et al., Biochemistry 28:4027 (1989); Kumar et al., in Cytoskeletal Proteins in Tumor Diagnosis, 1989, Weber et al., Eds., Cold Spring Harbor Press, p. 91!. Revertants of such transformed cells show normal levels of MLC-2 gene expression.
In view of the diverse roles played by oncogenes in cellular regulation and the relationship of oncogene activity to diseases such as cancer, it would be desirable to identify agents that can specifically alter oncogene-mediated biological processes, thereby reversing or suppressing the disease state. There is thus a need for specific in vitro screening systems for that purpose.
The proliferation and differentiation of mammalian cells are controlled by a family of polypeptide growth factors Holley, Nature 258:487 (1975)!. All polypeptide growth factors act by binding to specific cell surface receptors that, upon activation, transduce a broad range of signals leading to cell growth and differentiation James et al., Ann. Rev. Biochem. 53:259 (1984)!. A number of growth factors and their receptors have been characterized in recent years, including, e.g., epidermal growth factor (EGF), fibroblast growth factors (FGFs), platelet-derived growth factor (PDGF; a dimeric protein consisting of two "A" chains, two "B" chains or one "A" chain and one "B" chain), insulin-like growth factors (IGFs) and Bombesin. Many of the growth factor receptors have an intrinsic tyrosine kinase activity and contain very closely related structural elements.
Each growth factor may have a specificity for certain cells or tissue types. In many cases, however, they can also induce a response in other cell types. For example, EGF, the major target of which is epithelial cells, can also elicit a response from fibroblast cells. Fibroblast growth factor (FGF) is a potent stimulator of vascular endothelium and thus may be important in angiogenesis. At the same time FGF can stimulate other cell types such as fibroblasts and smooth muscle cells. PDGF is a key mitogen for smooth muscle cells and fibroblasts but has no direct effect on vascular endothelium or epithelium.
It has long been known that transformed cells in culture are generally able to grow in much lower concentrations of serum than are nontransformed cells. Serum is the normal source of growth factors for cultured cells. It was later discovered that fibroblasts transformed by certain retroviruses secrete factors which transiently induce normal cells to express a transformed phenotype Todaro et al., in Genes and Proteins in Oncogenesis, 1983, Weinstein and Vogel, Eds. Academic Press., New York, N.Y., pp. 165-181; DeLarco et al., Proc. Natl. Acad. Sci. USA 75:4001 (1978); Todaro et al., Cancer Res. 38:4147 (1978)!.
These factors, known as transforming growth factors (TGFs), consist of two functionally and structurally distinct groups of factors called TGF-.alpha. and TGF-.beta. Sporn et al., Nature 313:745 (1985)!. TGF-.beta. acts as a growth inhibitor for certain cell types, and as a mitogen for other cell types. The discovery of these TGFs led to the suggestion that one of the ways by which cells become transformed is by endogenous production of growth factors for which they have receptors Sporn et al., N. Eng. J. Med. 303:878 (1980)!. This internal production of growth factors is believed to serve as a constant stimulus for continued cell division, releasing the cells from their normal endogenous physiological controls.
The binding of growth factors to cellular receptors stimulates an array of biochemical responses, including changes in ion fluxes, activation of a number of protein kinases and alternation of transcriptional rates of several genes. These events culminate hours later in DNA replication and cell division. Recent studies have led to the delineation of pathways by which signals, generated at the membrane by the binding of a growth factor to its receptor, are transduced to the nucleus Ullrich et al., Cell 61:203 (1990); Williams, Science 24:1564 (1989)!. Increased expression of genes encoding transcription factors is an important element of the signal transduction mechanism which assures long term transcriptional response of cells to growth factors.
Smooth muscle .alpha.-actin isoform is expressed in both vascular smooth muscle and fibroblast cells Vandekerckhove et al., Differentiation 14:123 (1979); Leavitt et al., Nature 316:840 (1985)!. Actively proliferating aortic smooth muscle cells are known to contain relatively low levels of .alpha.-actin protein, whereas post-confluent cells show a nearly three-fold increase Owens et al., J. Cell Biol. 102:343 (1988); Corjay et al., J. Biol. Chem. 264:10501 (1989)!. Addition of PDGF to quiescent aortic smooth muscle cells results in a decrease in the steady state level of .alpha.-actin mRNA (Corjay et al., supra).
Abnormal cell proliferation due to the action of various growth factors is associated with a number of diseases such as neoplasia, atherosclerosis and myelofibrosis. To alleviate these conditions, it would be desirable to identify agents that can antagonize the actions of the responsible growth factors.
One of the most direct approaches to the identification of growth factor antagonists has entailed the use of assays based upon the binding of radiolabeled ligands to cellular receptors. Such assay systems are quite laborious and time consuming, however, and determination of the specificity of a given antagonist requires the use of a number of different radiolabeled growth factors and membrane receptor preparations.
An even more serious drawback to such assays is that they can detect only antagonists which act at the receptor level and interfere with growth factor binding. As noted above, however, a complex sequence of events occurs after a growth factor binds to its receptor. Intervention at multiple points by appropriate antagonists may thus be possible, but antagonists acting at points other than at the receptor cannot be identified by radioligand/receptor assays.
There is therefore a need for a more broadly-based growth factor antagonist screen that could identify a much wider range of antagonists, regardless of their locus of action.